Method and composition for antiviral therapy

ABSTRACT

A method of treatment of diseases of viral origin is disclosed. The method comprises oral or parenteral administration of an antiviral amount of a naturally occurring secoiridoid from plants of the family Oleaceae or derivatives thereof. Preferred oral dosage forms include the secoiridoid oleuropein in pure form or as a component of dried plant material of Olea europaea or a dried extract thereof and a pharmaceutically acceptable carrier.

This Application is a continuation of application Ser. No. 08/335/138filed Nov. 7, 1994 now abandoned.

FIELD OF THE INVENTION

This invention relates to treatment of disease of viral origin inwarm-blooded vertebrates. More particularly, this invention is directedto the use of secoiridoid compounds naturally occurring in plants of thefamily Oleaceae and derivatives thereof.

BACKGROUND AND SUMMARY OF THE INVENTION

The olive tree and other members of the Family Oleaceae have beendocumented as a source of medicinal substances since biblical times.Needless to say, many researchers have studied the cocktail ofphytogenic substances produced by the olive and other members of theFamily Oleaceae. One compound that has received attention from theresearch community is the secoiridoid glucoside oleuropein, a compoundof the formula ##STR1## wherein R is glycosyl and R₁ is2-(3,4-dihydroxyphenyl) ethyl. Related secoiridoids wherein R₁ is H, CH₃or 2-(4-hydroxyphenyl ethyl) are also known to be endogenous to manyplant species of the family Oleaceae, although in lesser concentrationsand in fewer identified species than the ubiquitous oleuropein.

Animal studies have revealed that oleuropein itself or as a component ofextracts of plant tissues containing that compound exhibit bothhypoglycemic and cardiovascular effects. It is also known thatoleuropein can be acid hydrolyzed to produce (-)-elenolic acid, acompound which has been reported to have antiviral properties in vitro,but little, if any, activity in vivo.

The present invention is based on the discovery that secoiridoidglucosides of the formula ##STR2## wherein R is glycosyl and R₁, ishydrogen or an ester-forming group are metabolized in vivo to thedextrorotatory form of elenolic acid [(+)-elenolic acid], a compound ofthe formula ##STR3## wherein R₁, is hydrogen, a compound which isbelieved to be more available in vivo than the correspondingdiasteromer(-)-elenolic acid of the formula ##STR4## The enhanced invivo efficacy of the oleuropein metabolite, (+)-elenolic acid relativeto the corresponding levorotatory compound is thought to be due, atleast in part, to its reduced affinity for serum proteins and thus itsgreater availability for uptake by virus infected tissues. Antivirallyeffective blood levels of (+)-elenolic acid can also be achieved byadministration of (+)-elenolic acid and its esters of the formula##STR5## wherein R₁ is hydrogen or a pharmaceutically acceptableester-forming group and salts thereof, which can be prepared fromsecoiridoid glucosides naturally occurring in plant material of thefamily Oleaceae via extraction and controlled enzyme (glucosidase)hydrolysis and/or deesterification/transesterification reactions.

Thus, it is one object of the present invention to provide a method oftreatment of disease of viral origin in warm-blooded vertebrates byadministering antiviral compositions containing secoiridoid glucosidesnative to the plant family Oleaceae and derivatives thereof.

Another object of the invention is to provide oral dosage forms ofsecoiridoid glucosides of Olea europaea and derivatives thereof.

In another more particular aspect of this invention plant material ofthe family Oleaceae and extracts thereof containing naturally occurringoleuropein glucosides and enzyme hydrolysates thereof are administeredin treatment of diseases of viral origin in warm-blooded vertebratessuffering from such diseases.

One further object of this invention is a method for establishingantivirally effective blood levels of (+)-elenolic acid by theadministration of oleuropein glucosides native to the family Oleaceae ortheir derivatives via transesterification, diesterification and/orglucolysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of treatment of disease ofviral origin in warm-blooded vertebrates and to pharmaceuticalformulations for use in such treatment methods. The method comprises thestep of administering to a vertebrate suffering from a disease of viralorigin an antivirally effective amount of an antiviral compositioncomprising a compound of the formula ##STR6## and a pharmaceuticallyacceptable carrier therefor. In the above formula, the group R isglucosyl and R₁, is hydrogen or a pharmaceutically acceptableester-forming group. When R₁ is hydrogen, the acid compound representedcan be utilized in the form of one of its pharmaceutically acceptablesalts.

There are many diseases of viral etiology that afflict man and animal.In man, diseases such as hepatitis, mononucleosis, shingles, herpes,influenza, the common cold and even certain types of leukemia are knownto be of viral etiology. Viral infections are also common in many animalspecies, both in meat producing and in companion animals. Rotovirusinfections plague swine. Cattle develop bovine rhinovirus infections(shipping fever) when subjected to conditions of stress. Canineparvovirus and feline leukemia virus are common viral infections inthose companion animals species. Such diseases of viral origin can betreated with resultant reduction in clinical symptomology by therapeuticadministration of antiviral compositions in accordance with thisinvention.

The antiviral compositions administered in accordance with thisinvention comprise a compound of Formula I or II above in combinationwith a pharmaceutically acceptable carrier. The compounds of Formula Iwherein R₁ is 2-(4-hydroxyphenyl)ethyl or 2-(3,4-dihydroxyphenyl)ethylare naturally occurring compounds in many plants of the Family Oleaceae,including members of the genus Fraxinus, Syringa and the genusLigustrum. Preferred plant sources of the naturally occurringsecoiridoids of Formula I wherein R₁ is 2-(4-hydroxyphenyl)ethyl and2-(3,4-dihydroxyphenyl)ethyl are varieties of Olea europaea (the olive).Preferred varieties of Olea europaea as a source of secoiridoidglycosides for use in accordance with this invention are the varietiesManzanillo and Mission.

The most prevalent of the secoiridoid compounds in such varieties is thecompound of Formula I wherein R₁, is 2-(3,4-dihydroxyphenyl)ethyl and Ris glucosyl, a compound given the common name oleuropein. That compoundcan be readily isolated from plant material, preferably ground leaves ofthe olive by aqueous or aqueous-alcoholic extraction at room temperatureor above, preferably at elevated temperature of about 40 to about 100°C.

Oleuropein can then be purified, for example, by chromatographicseparation procedures. That compound can then be used to formulateantiviral compositions in accordance with this invention or to prepareother antivirally effective compounds represented by Formulas I or II.Thus, for example, oleuropein can be subjected, to base catalyzedtransesterification wherein the R₁, group 2-(3,4-dihydroxyphenyl)ethylis exchanged with another pharmaceutically acceptable ester-forminggroup. The term "pharmaceutically acceptable ester-forming group" asused in defining the present invention, refers to those ester-forminggroups which when cleaved via esterase reactions in vivo producesubstantially non-toxic, physiologically compatible alcohols. Suitablepharmaceutically acceptable ester-forming groups include C₁ -C₈ loweralkyl, and substituted C₁ -C₈ alkyl, benzyl, substituted benzyl whereinthe substituents are halo, C₁ -C₄ alkoxy, C₁ -C₄ acyloxy, and the like.The compound of Formula I wherein R₁, is hydrogen can be produced byesterase-mediated deesterification of oleuropein, typically in anaqueous medium at a pH between about 6 and about 8.5.

The compounds of Formula II are prepared from the correspondingcompounds of Formula I by treatment with glucosidase, preferably thatfrom the olive at a pH of about 4 to about 5. The compound of Formula IIwherein R₁ is hydrogen is (+)-elenolic acid.

The compounds of Formula I or II wherein R₁ is hydrogen representcarboxylic acids and such acids can be used in accordance with thisinvention in the acid form or in the form of their pharmaceuticallyacceptable salts formed with organic bases or inorganic bases, such asammonium, alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Bases useful in preparing such salts includesodium hydroxide, potassium hydroxide, ammonium hydroxide, and potassiumcarbonate. Of the salt forms, the potassium and sodium salts areparticularly preferred.

The antiviral compositions of the present invention can be administeredorally or parenterally in an antivirally effective amount to treat,i.e., reduce the symptoms, of diseases of viral origin. Oral dosageforms can be in a solid or liquid form and comprise an antivirallyeffective amount of a compound of Formula I or Formula II above and apharmaceutically acceptable carrier. Such dosage forms can be formulatedfrom pure compounds of Formula I or Formula II, or they can beformulated from ground plant materials of the family Oleaceae,preferably leaves of Olea europaea, or aqueous or aqueous alcoholicextracts thereof. Thus, for example, extraction of dried olive leaveswith two volumes of a 12-15% ethanol/water solution for 10 days at roomtemperature provides an extract containing about 70 to about 250 mg ofoleuropein per two ounces of the liquid extract. The extract itself canbe administered orally as an antiviral composition in accordance withthe method of treatment of the present invention, or aqueous or aqueousalcoholic (preferably methanol or ethanol) extracts can be spray-driedto provide a dry powder which can be formulated into oral dosage formswith other pharmaceutically acceptable carriers.

The solid oral dosage form compositions in accordance with thisinvention are prepared in a manner well known in the pharmaceutical art,and comprise at least one compound of Formula I or Formula II associatedwith at least one pharmaceutically acceptable carrier. In making suchcompositions, the compound of Formula I or Formula II, either in pureform or as a component of ground plant material or extracts thereof, areusually mixed, diluted or enclosed within a carrier. The carrier can bein a solid form, semi-solid or liquid material which acts as a vehicle,carrier or medium for the active ingredient. Alternatively, the carriercan be in the form of a capsule or other container to facilitate oraladministration. Thus the solid oral dosage forms for administration inaccordance with the method of this invention can be in the form oftablets, pills, powders or soft or hard gelatin capsules. Alternatively,the antiviral compositions in accordance with this invention for oraladministration can be in liquid form wherein the pharmaceuticallyacceptable carrier is water or an aqueous alcoholic medium. Thecompositions for administration in the present method can also beformulated with other common pharmaceutically acceptable excipients,including lactose, dextrose, sucrose, sorbitol, mannitol, starches,gums, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, methylcellulose, water, alcohol and the like. Theformulations can additionally include lubricating agents such as talc,magnesium stearate and mineral oil, wetting agents, emulsifying andsuspending agents, preserving agents such as methyl- andpropylhydroxybenzoates, sweetening agents or flavoring agents. Furtherthe compositions of the present invention can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the patient by employing procedures well knownin the art.

Orally administered compositions are preferably formulated in unitdosage form with each dosage normally containing from about 30 to about500 mg of a compound of the Formula I or Formula II, more typicallyabout 100 to about 500 mg of such active compounds. The term "unitdosage form" refers to physically discrete units suitable as unitarydosages for human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with a suitable pharmaceuticalcarrier/excipient. The preferred dosage levels of the compounds ofFormula I/Formula II for treatment of viral infections in accordancewith this invention depend on the route of administration, the nature ofthe active compound or combination of active compounds, and patientcondition. When administered orally for treatment of viral disease, thecompounds of Formula I or Formula II are administered at a dose of about0.1 to about 15 mg/kg, more preferably about 0.2 to about 10 mg/kg ofpatient/animal body weight. In treatment of viral infections oral dosageforms in accordance with this invention can be administered 1 to 4 timesa day, again depending on patient condition and the nature of thedisease being treated.

Similar considerations bear on the dosage range for parenteraladministration of compounds of Formula I or Formula II in treatment ofviral infections in accordance with this invention. Parenteral dosesare, however, typically lower than those required for antiviral efficacyvia the oral route of administration. Thus, antiviral treatment can beachieved by parenteral administration of about 0.05 to about 3 mg/kg ofpatient/animal body weight. Parenteral formulations for use inaccordance with the present invention are prepared using standardtechniques in the art. They are commonly prepared as sterile injectablesolutions, using a parenterally acceptable carrier such as isotonicsaline solution or as a sterile packaged powder prepared forreconstitution with sterile buffer or isotonic saline prior toadministration to a patient. The injectable formulation can contain fromabout 1 to 50 mg of a compound of Formula I or II per ml of formulation.

Administration of an antivirally effective amount of a compositioncomprising a compound of Formula I or Formula II and a pharmaceuticallyacceptable carrier in accordance with this invention produce antivirallyeffective blood levels of (+)-elenolic acid, a compound of the formula##STR7## via a heretofore unappreciated in vivo hydrolysis, and in thecase of the compounds of Formula I, stereoselectivehydrolysis/rearrangement. Administration of antiviral compositions of acompound of Formula I as part of ground/dried native plant material,preferably olive leaves, or as an aqueous or alcoholic extract of oliveleaves is a particularly preferred embodiment in accordance with thisinvention. While not wishing to be bound by theory, the level ofantiviral activity associated with such antiviral compositions mayderive from a synergistic antiviral effect with other natural componentsof the olive leaf, such as the flavonoids rutin, hesperidin, andluteolin-7-glucoside.

The following non-limiting examples are illustrative of the method andcompositions of the present invention. It is understood, however, thatsuch examples are but representative of various embodiments of theinvention and it is not intended that the invention be limited to thescope of the examples.

EXAMPLE 1

(A) A volume of dried leaves of Olea europaea is suspended in 2 volumesof red wine and held at room temperature for about 7 to about 10 dayswith periodic stirring. Filtration of the mixture provides a tincturecontaining about 88 mg of oleuropein per ounce of fluid.

(B) A volume of dried leaves of Olea europaea is suspended in twovolumes of water and the resulting suspension is then subjected toconditions of high shear in a Waring blender to produce a dispersion offinely divided plant material which was held at a temperature of about40 to about 65° for two days. Filtration of the mixture provides anaqueous extract containing about 72 mg of oleuropein per ounce of fluid.The aqueous extract is optionally blended with effective amounts ofsweetening and/or flavoring agents to provide a palatable liquid oraldosage form of oleuropein.

(C) One volume of leaves or buds of Olea europaea is combined with abouttwo volumes of a 3:2 mixture of methanol and water. The resultingaqueous alcoholic suspension of plant material is heated for 16 hours atabout 75° C., cooled and filtered to provide an aqueous alcoholicextract. The extract is spray-dried to produce a powder comprisingoleuropein. The powder is filled into gelatin capsules in an amountsufficient to provide 30 to about 500 mg of oleuropein per capsule.

(D) The spray-dried extract of (C) above is subjected to high pressureliquid chromatography to produce oleuropein [Formula I; R=glycosyl; R₁=2-(3,4-dihydroxyphenyl)ethyl] and ligstroside [Formula I; R=glycosyl;R₁ =2-(4-hydroxyphenyl)ethyl] in substantially pure form. The purifiedoleuropein is formulated alone or in combination with ligstroside withtabletting starch and a tabletting lubricant, magnesium stearate, toform a tabletting mixture. The tabletting mixture is pressed intocompressed tablets containing about 30 to about 500 mg of oleuropein pertablet.

EXAMPLE 2

Oleuropein (1 g) is dispersed in 50 ml of methanol. The solution iscooled to about 10° C. and treated with stirring with about 2 g ofpotassium hydroxide pellets. The mixture is allowed to warm to roomtemperature and after about 6 hours, the reaction mixture is dilutedwith about 60 ml of 6 N HCl (pH about 7.5) and evaporated to dryness.The chromatographic purification of the product mixture providesoleoside [Formula I; R=glycosyl, R₁ =methyl]. The purified oleoside isformulated into a solid oral dosage form containing about 250 mg ofoleoside. Alternatively, oleoside is dissolved in sterile isotonicsaline at a concentration of about 5 mg per ml to provide a parenteraldosage form for use in accordance with the method of this invention.

EXAMPLE 3

Five grams of oleuropein is dissolved in 500 ml of water buffered at pH5.0 and treated with glucosidase until analysis of the reaction mixtureby thin layer chromatography indicates completion of the reaction.Standard workup of the reaction mixture followed by chromatographicpurification of the product mixture provides a compound of Formula IIwherein R₁ =2-(3,4-dihydroxyphenyl)ethyl. The purified product isformulated into oral or parenteral dosage forms and administered fortreatment of viral diseases in accordance with the method of the presentinvention.

EXAMPLE 4

A solution of 500 mg of the product of Example 3 in 50 ml of waterbuffered at about pH 7.5 to about 8.5 is treated with commerciallyavailable esterase at a temperature of about 30° C. until thin layerchromatographic analysis of the esterase reaction mixture indicatescompletion of the reaction due to disappearance of the startingcompound. After reaction completion, the pH of the mixture is readjustedto about 8.5 and after washing it twice with ethyl acetate, the pH ofthe solution is adjusted to about 4.5 in the presence of 30 ml of ethylacetate. The ethyl acetate acid extract of the reaction mixture isseparated, washed with distilled water and brine, and dried overanhydrous sodium sulfate. Evaporation to dryness provides (+)-elenolicacid [Formula II; R₁ =hydrogen] in substantially pure form. The productis further purified by chromatography and/or crystallization as itssodium or potassium salt. It is formulated into oral or parenteraldosage forms in accordance with this invention for treatment of viralinfections in warm-blooded vertebrates.

EXAMPLE 5

Dried leaves of Olea europaea are ground to a fine powder and loadedinto gelatin capsules such that each capsule contains a volume of groundplant material containing between about 30 and about 500 mg ofoleuropein per capsule. Dried olive leaves typically contain about 60 toabout 90 mg of oleuropein per gram of dried leaf. The capsules areadministered orally for treatment of diseases of viral origin inaccordance with this invention.

EXAMPLE 6

The antiviral efficacy of a composition of Example 1(A) above wasevaluated in treatment of six subjects afflicted with herpes virusinfections. Each of the six subjects ingested two ounces of theformulation every six hours. All subjects reported reduction of herpeticlesions. Three subjects reported disappearance of lesions in 36-48 hoursafter initiating treatment. One subject, a 34 year old Caucasian female,had several months earlier discontinued use of birth control pills andit was thought that her estrogen surges were causing immunosuppressionwhich complicated her infection. After three days of doubling the dosageof the tincture (four ounces every six hours) most of her lesions wereresolved. Two other subjects, a male and a female, more recentlyinfected were also given the higher doses (four ounces every six hoursorally) and each reported improvement measured by reduction in severityof their herpetic lesions, and in at least one patient, a 22.8% decreasein antibody titers (IgG) three weeks after initiating therapy with theoleuropein-containing olive leaf extract.

I claim:
 1. A method of treating a warm-blooded vertebrate sufferingfrom a disease of viral origin, said method comprising the step ofadministering orally or parenterally to said vertebrate atherapeutically effective amount of an antiviral composition comprisinga compound selected from the group consisting of oleuropein which is acompound of the formula ##STR8## wherein R₁ is2-(3,4-dihydroxyphenyl)ethyl and R is glucosyl, deesterified oleuropeinof Formula I above wherein R₁ is hydrogen, pharmaceutically acceptablesalts of deesterified oleuropein, and other esters of formula I whereinR₁ is derived from substantially non-toxic physiologically compatiblealcohols, and a pharmaceutically acceptable carrier therefor.
 2. Themethod of claim 1, wherein the compound is oleuropein.
 3. The method ofclaim 1, wherein the disease of viral origin is selected from the groupconsisting of herpes mononucleosis, hepatitis, and diseases derivingfrom infection by rotovirus, bovine rhinovirus, canine parvovirus andfeline leukemia virus.